The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They possess 5kb linear single-stranded DNA (ssDNA) genomes and their replication relies extensively on host nuclear factors. Our recent work has shown how the expression profiles of many of the parvoviruses feature impressively intricate processes of transcriptional transactivation, alternative RNA processing, and alternative translation initiation, which maximize their coding capacity. We have identified three aspects critical for parvovirus replication to pursue in this application. 1. Unlike AAV2, AAV5 Rep-encoding P19-generated transcripts are polyadenylated within the central intron and therefore not spliced, precluding an AAV2-like mechanism for making both Rep 40 and Rep52. Surprisingly, AAV5 ensures the production of a Rep 40-like protein by utilizing a new internal translation initiation event. The 10 nt RNA region lying between the two AUGs of AAV5 functions as a novel, positively-acting signal to program internal initiation. This different AAV5 expression strategy also generates a Rep 40 protein with a very different structure than that produced by AAV2. Thus, we propose in Specific Aim I: To characterize alternative translation initiation of the AAV small rep gene and it protein products. 2. Translation of the goose parvovirus (GPV) spliced rep gene mRNA initiates internally, specifically bypassing a strong initiating AUG used in unspliced RNA. Although the internal initiation choice occurs in the cytoplasm during translation, it is dictated by elements within the GPV intron not present in the translated mRNA. Alternative translation initiation of the rep-gene is directed by the splicing process itself, and by the nature of the intervening intron, and thus nuclear processing of GPV RNAs influences the translation fate of these RNAs in the cytoplasm. Therefore, we propose in Specific Aim II: To characterize how nuclear RNA processing events govern alternative translation of GPV mRNAs. 3. The capsid gene of minute virus of canines (MVC) contains a potent internal polyadenylation site, and we have recently discovered that the MVC NP1 protein is essential for the accumulation in the cytoplasm of partially-processed MVC capsid gene mRNAs that retain that site, and thus for the production of the capsid proteins. This represents a novel way in which this parvovirus regulates access to capsid gene information. Additionally, NP1 is the first parvovirus protein shown to be directly involved in RNA processing. Thus, we propose in Specific Aim III: To characterize the function of NP1, a unique viral RNA processing factor. These studies will clarify critical aspects of parvovirus gene expression and will develop new paradigms in the field. Our systems afford highly tractable, tightly controlled models that are positioned to both advance parvovirology and to deliver significant insight into fundamental aspects of gene expression.